1. Field of the Invention
The present invention relates to methods for the analysis of nucleic acids and the identification of genotypes present in biological samples. More specifically, embodiments of the present invention relate to automated methods for genotyping and analyzing the sequences of nucleic acids.
2. Description of Related Art
The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. PCR is perhaps the most well-known of a number of different amplification techniques.
PCR is a powerful technique for amplifying short sections of DNA. With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated so that there are enough copies to be detected and analyzed. In principle, each cycle of PCR could double the number of copies. In practice, the multiplication achieved after each cycle is always less than 2. Furthermore, as PCR cycling continues, the buildup of amplified DNA products eventually ceases as the concentrations of required reactants diminish. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).
Real-time PCR refers to a growing set of techniques in which one measures the buildup of amplified DNA products as the reaction progresses, typically once per PCR cycle. Monitoring the accumulation of products over time allows one to determine the efficiency of the reaction, as well as to estimate the initial concentration of DNA template molecules. For general details concerning real-time PCR see Real-Time PCR: An Essential Guide, K. Edwards et al., eds., Horizon Bioscience, Norwich, U.K. (2004).
More recently, a number of high throughput approaches to performing PCR and other amplification reactions have been developed, e.g., involving amplification reactions in microfluidic devices, as well as methods for detecting and analyzing amplified nucleic acids in or on the devices. Thermal cycling of the sample for amplification in microfluidic devices is usually accomplished in one of two methods. In the first method, the sample solution is loaded into the device and the temperature is cycled in time, much like a conventional PCR instrument. In the second method, the sample solution is pumped continuously through spatially varying temperature zones. See, e.g., Lagally et al. (Analytical Chemistry 73:565-570 (2001)), Kopp et al. (Science 280:1046-1048 (1998)), Park et al. (Analytical Chemistry 75:6029-6033 (2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S. Pat. No. 6,960,437) and Knapp et al. (U.S. Patent Application Publication No. 2005/0042639).
Once there are a sufficient number of copies of the original DNA molecule, the DNA can be characterized. One method of characterizing the DNA is to examine the DNA's dissociation behavior as the DNA transitions from double stranded DNA (dsDNA) to single stranded DNA (ssDNA). The process of causing DNA to transition from dsDNA to ssDNA with increasing temperature is sometimes referred to as a “high-resolution temperature (thermal) melt (HRTm)” process, or simply a “high-resolution melt” process. Alternatively, the transition from ssDNA to dsDNA may be observed through various electrochemical methods, which generate a dynamic current as the potential across the system is changed.
Melting profile analysis is an important technique for analyzing nucleic acids. In some methods, a double stranded nucleic acid is denatured in the presence of a dye that indicates whether the two strands are bound or not. Examples of such indicator dyes include non-specific binding dyes such as SYBR® Green I, whose fluorescence efficiency depends strongly on whether it is bound to double stranded DNA. As the temperature of the mixture is raised, a reduction in fluorescence from the dye indicates that the nucleic acid molecule has melted, i.e., unzipped, partially or completely. Thus, by measuring the dye fluorescence as a function of temperature, information is gained regarding the length of the duplex, the GC content or even the exact sequence. See, e.g., Ririe et al. (Anal Biochem 245:154-160, 1997), Wittwer et al. (Clin Chem 49:853-860, 2003), Liew et al. (Clin Chem 50:1156-1164 (2004), Herrmann et al. (Clin Chem 52:494-503, 2006), Knapp et al. (U.S. Patent Application Publication No. 2002/0197630), Wittwer et al. (U.S. Patent Application Publication No. 2005/0233335), Wittwer et al. (U.S. Patent Application Publication No. 2006/0019253), Sundberg et al. (U.S. Patent Application Publication No. 2007/0026421) and Knight et al. (U.S. Patent Application Publication No. 2007/0231799).
An alternative method for analyzing a nucleic acid uses voltammetry to detect electrochemical biosensors to detect nucleic acid hybridization. Electrochemical technology is miniaturizable, accurate, and sensitive with controlled reaction conditions. Both label-free and labeled approaches exist for detecting nucleic acid hybridization. Label-free approaches generally rely on changes to the electrical properties of an interface when bound to a nucleic acid, changes in flexibility between rigid dsDNA and more flexible ssDNA, or electrochemical oxidation of guanine bases. See, e.g., Gooding (Electroanalysis 14:1149-1156, 2002), Gooding et al. (Chem. Commun. 2003:1938-1939, 2003), Mearns et al. (Electroanalysis 18:1971-1981, 2006); Paleck (Electroanalysis 8:7-14, 1996). Labeled approaches for detecting nucleic acid hybridization are more common and well-known than label-free approaches. These approaches generally involve redox active molecules that intercalate between Watson-Crick base pairs of a nucleic acid or in the minor or major grooves of the nucleic acid secondary structure, and thus do not interact with single-stranded nucleic acids. Examples of such redox active molecules include Co(Phen)33+, Co(bpy)33+, and Methylene Blue. See, e.g., Mikkelsen (Electroanalysis 8:15-19, 1996); Erdem et al. (Anal. Chim. Acta 422:139-149, 2000). In some cases, the redox active molecules bind preferentially to either dsDNA or ssDNA. Another alternative method includes attaching a label group, such as a ferrocene group, to the end of a nucleic acid probe, which is immobilized on an electrode surface. See, e.g., Mearns et al. (Electrochemistry 18:1971-1981, 2006); Anne et al. (J. Am. Chem. Soc. 128:542-547, 2006); Lai et al. (Proc. Natl. Acad. Sci. U.S.A. 103:4017-4021, 2006); Fan et al. (Proc. Natl. Acad. Sci. U.S.A. 100:9134-9147, 2003); Xiao et al. (Proc. Natl. Acad. Sci. U.S.A. 103:16677-16680, 2006). The single-stranded probe molecule is flexible enough that the ferrocene group may come within close enough contact with the electrode surface to be oxidized or reduced. However, upon hybridization, the rigid double-stranded nucleic acid molecule stands normal to the electrode surface, and the ferrocene group is sufficiently far from the electrode that it will not be oxidized or reduced.
These systems may all be interrogated through cyclic voltammetry. By applying an electric potential that increases or decreases over time across the system, a variable electric current is generated as the label or DNA molecule is oxidized or reduced. Complete hybridization of the target molecule to the probe molecule will generate a characteristic dynamic profile of current generated versus voltage applied. Incomplete hybridization, which would occur if the target molecule contained a mutant genotype, would result in a differing dynamic profile of current generated versus voltage applied. Thus, different nucleic acid sequences may be distinguished from one another through examination of their respective voltammograms.
Some nucleic acid assays require differentiation between potential genotypes within a class of known genotypes. Generally, for thermal melt analysis, researchers will visually inspect a thermal melt profile to determine the melting temperature of the nucleic acid in the sample. However, some nucleic acid assays require identification of a single nucleotide change where the difference in melting temperature (Tm) between the wild type nucleic acid and a mutant nucleic acid is quite small (e.g. less than 0.25° C.). This level of temperature resolution is difficult to achieve in a visual inspection. Furthermore, visual inspection of thermal melt profiles to determine melting temperature ignores significant additional information contained in the profiles, such as the overall shape and distribution of the profile.
Accordingly, what are desired are methods and systems for high resolution melt analysis that are capable of more accurately discriminating thermal melt curves and obtaining DNA sequence information from these melting curves, especially where these thermal melt curves are differentiated by a small temperature range. Also desired are methods and systems for high resolution melt analysis that more accurately identify thermal melt curves that facilitate detection of sequence information for DNA that contain one or more peaks or mutations. Also desired are methods and systems for that are capable of more accurately identifying a nucleic acid sequence and discriminating between similar sequences while taking into account both features of the profile as well as the overall shape. Also desired are methods that are capable of rapidly identifying a genotype with minimal intervention and decision-making from the user.